Handbook of Size Exclusion Chromatography and Related ...

The use of freshly distilled organic solvents is always recommended.
For aqueous mobile phases, the use of Nanopure TM (Barnstead International,
Dubuque, Iowa, U.S.A.) or equivalently purified and filtered water must be used.
Particular care must be exercised in preparing the buffered solutions so often
required for various types of biopolymers. Despite the high purity listed on
containersofthesefinechemicals,rarelyistherementionmadeofthedustcontent
of the ingredients. Thus such mobile phases must be filtered with great care
throughout their preparation.
Finally,the sample itself may contain large quantities of extemporaneous
debris introduced during sample preparation. For this reason, aguard column is
often used to protect the columns from the clogging that such debris may cause.
For certain types of separation mechanisms, such as asymmetric flow field flow
fractionation (AsFFF), the debris is often removed directly by the separation
process itself (15).
The columns shown in Fig. 2generally refer to SEC columns, although the
MALS measurement is independent of the separation (or nonseparation) method.
Reversed phase HPLC columns are often used instead of the SEC columns,
especiallyforthemeasurementofproteins.Forthesemeasurements,theDRIdetector
is generally replaced by aUV detector because most DRI detectors do not have the
dynamicrangeneededtocopewiththerefractiveindexrangeofthemobilephase.As
mentioned earlier,aguard column is often included for many such separations.
Inrecentyears,thedevelopmentofmorerobustinstrumentationforthefield
flow fractionation method of separation has permitted the incorporation of such
instrumentation without the steep learning curve associated historically with its
implementation. The AsFFF device alluded to earlier and introduced by Wyatt
Technology Europe as the Eclipse TM (Woldert, Germany) is aparticular case in
point. For many polymers, particularly water-soluble polymers, the separations
achieved rival SEC. Mastery of the device can be achieved within afew hours.
This should be compared with weeks or months formerly required to learn the
subtleties of such separation devices. In addition to so-called “cross-flow” FFF
(exemplified by the AsFFF devices), there are several other FFF separation
techniques(16) based on thermal, centrifugal,electrical,orother propertiesofthe
molecules undergoing separation. Acurrent reference list of all articles published
in the field of FFF may be found at the website developed by Dr. Mark Shure of
Rohm and Haas (http://www.rohmhaas.com/fff/).
The FFF separation techniques are particularly useful for the separation of
nanoparticles and giant molecules such as DNA. SEC separations, while generally
inapplicable to particles, have been used for many years to separate (or attempt to
separate) large molecules, often beyond the exclusion limit. Unfortunately, such
molecules tend to shear during separation, which results in a distribution of
molecules separated that includes often substantial amounts of such fragmented
contributions.
© 2004 by Marcel Dekker, Inc.